Wnt Pathway Antagonists

ABSTRACT

Aromatic compounds for treating various diseases and pathologies are disclosed. The methods use of such compounds are also provided. Accordingly, the present invention makes available methods and compositions for inhibiting aberrant growth states in cells having Wnt receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)of U.S. U.S. Ser. No. 60/507,163, filed Sep. 29, 2003, the entirecontent of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates generally to the use of chemical compoundsto treat a variety of disorders, diseases and pathologic conditions andmore specifically to the use of various aromatic compounds forinhibiting signaling pathways.

BACKGROUND INFORMATION

Pattern formation is the activity by which embryonic cells form orderedspatial arrangements of differentiated tissues. Speculation on themechanisms underlying these patterning effects usually centers on thesecretion of a signaling molecule that elicits an appropriate responsefrom the tissues being patterned. More recent work aimed at theidentification of such signaling molecules implicates secreted proteinsencoded by individual members of a small number of gene families.

A longstanding idea in cancer biology is that cancers arise and grow dueto the formation of cancer stem cells, which may constitute only aminority of the cells within a tumor but are nevertheless critical forits propagation. Stem cells are appealing as the cell of origin forcancer because of their pre-existing capacity for self-renewal and forunlimited replication. In addition, stem cells are relatively long-livedin comparison to other cells within tissues, providing a greateropportunity to accumulate the multiple additional mutations that may berequired to increase the rate of cell proliferation and produceclinically significant cancers. Of particular recent interest in theorigin of cancer is the observation that the Wnt signaling pathway,which has been implicated in stem cell self-renewal in normal tissues,upon continuous activation has also been associated with the initiationand growth of many types of cancer. This pathway thus provides apotential link between the normal self-renewal of stem cells and theaberrantly regulated proliferation of cancer stem cells.

The Wnt growth factor family includes more than 10 genes identified inthe mouse and at least 7 genes identified in the human. Members of theWnt family of signaling molecules mediate many important short- andlong-range patterning processes during invertebrate and vertebratedevelopment. The Wnt signaling pathway is known for its important rolein the inductive interactions that regulate growth and differentiation,and likely also plays important roles in the homeostatic maintenance ofpost-embryonic tissue integrity. Wnt stabilizes cytoplasmic β-catenin,which stimulates the expression of genes including c-myc, c-jun, fra-1,and cyclin D1. In addition, mis-regulation of Wnt signaling can causedevelopmental defects and is implicated in the genesis of several humancancers. More recently, the Wnt pathway has been implicated in themaintenance of stem or progenitor cells in a growing list of adulttissues that now includes skin, blood, gut, prostate, muscle and thenervous system.

Thus, portions of the Wnt pathway can be taken as target molecules forthe regulation of cell growth, oncogenesis and apoptosis of cells. Theability to modulate activity of the Wnt signaling pathway represents apossible therapeutic approach to several clinically significant cancers.A need therefore exists for methods and compounds that inhibit signaltransduction activity by modulating activation of a Wnt-mediated signaltransduction pathway, to reverse or control aberrant growth.

SUMMARY OF THE INVENTION

The present invention makes available methods and reagents, involvingcontacting a cell with an agent, such as an aromatic compound, in asufficient amount to antagonize a Wnt activity, e.g., to reverse orcontrol an aberrant growth state.

According to one embodiment of the invention, the compound havingstructure (I), or a pharmaceutically acceptable salt thereof, isprovided:

According to another embodiment of the invention, the compound havingstructure (II), or a pharmaceutically acceptable salt thereof, isprovided:

According to another embodiment of the invention, the compound havingstructure (II), or a pharmaceutically acceptable salt thereof, isprovided:

According to yet another embodiment of the invention, compoundsincluding a first aromatic moiety fused to a second aromatic moiety areprovided, wherein the first aromatic moiety is naphthalene-1,4-dionemoiety and the second aromatic moiety is an N-substitutedtriazole-N-oxide moiety. An example of the substitutent that can be usedin the N-substituted triazole-N-oxide moiety includes an alkylarylgroup, such as benzyl group.

According to yet another embodiment of the invention, compoundsincluding a benzopiperazine moiety fused to a substituted imidazolemoiety are provided. The benzopiperazine moiety can include analkylpiperazinyl group, such as methylpirazinyl group. The imidazolemoiety can include a phenyl substituent, and can further include ahalogenated aromatic group, e.g., a bromophenyl group, attached to anitrogen atom in the imidazole structure.

According to yet another embodiment of the invention, compoundsincluding an azaanthracene moiety and a secondary amino moiety, or apharmaceutically acceptable salt thereof, are provided. The secondaryamino moiety can be attached to the nitrogen-containing ring of theazaanthracene moiety.

According to another embodiment of the invention, a method for treatinga disorder is provided, the method can include administering aneffective amount of a compound according to any embodiment of thepresent invention, or any combination thereof, or pharmaceuticallyacceptable salts, hydrates, solvates, crystal forms and individualdiastereomers thereof, to a subject in need of such treatment. Thecompound can be administered in combination with a therapeutic agent,immunomodulatory agent, therapeutic antibody or an enzyme inhibitor.

Thus, in one embodiment, the methods of the present invention includethe use of aromatic compounds that agonize inhibition of Wnt signaling,such as by inhibiting activation of Wnt downstream components of thesignaling pathway, in the regulation of repair and/or functionalperformance of a wide range of cells, tissues and organs, includingnormal cells, tissues, and organs. For instance, the subject method hastherapeutic and cosmetic applications ranging from regulation of neuraltissues, bone and cartilage formation and repair, regulation ofspermatogenesis, regulation of smooth muscle, regulation of lung, liverand other organs arising from the primitive gut, regulation ofhematopoietic function, regulation of skin and hair growth, etc.Moreover, the subject methods can be performed on cells that areprovided in culture (in vitro), or on cells in a whole animal (in vivo).

In another embodiment, the subject method can be used to treatepithelial cells having a Wnt receptor. For instance, the subject methodcan be used in treating or preventing basal cell carcinoma or other Wntpathway-related disorders. In certain embodiments, a subject antagonistmay inhibit activation of a Wnt pathway by binding to patched.

In another embodiment, the present invention provides pharmaceuticalpreparations comprising, as an active ingredient, a Wnt antagonist, suchas described herein, formulated in an amount sufficient to inhibit, invivo, proliferation or other biological consequences of Wntmis-expression.

In another embodiment, the subject method can be used as part of atreatment regimen for malignant medulloblastoma and other primary CNSmalignant neuroectodermal tumors. In other embodiments, the subjectmethod can be used as part of a treatment regimen for rhabdomyosarcoma,lung cancer, and in particular small cell lung cancer, gut-derivedtumors, including but not limited to cancer of the esophagus, stomach,pancreas, and biliary duct system; prostate and bladder cancers, coloncancer, or liver cancer.

The subject treatments using Wnt antagonists can be effective for bothhuman and animal subjects. Animal subjects to which the invention isapplicable extend to both domestic animals and livestock, raised eitheras pets or for commercial purposes. Examples are dogs, cats, cattle,horses, sheep, hogs, and goats.

According to another embodiment of the invention, a pharmaceuticalcomposition is provided. The composition can include a compoundaccording to any embodiment of the present invention, or any combinationthereof, in a pharmaceutically acceptable carrier.

According to another embodiment of the invention, an article ofmanufacture is provided, the article comprising packaging material and apharmaceutical composition contained within the packaging material,where the packaging material comprises a label which indicates that thepharmaceutical composition can be used for treatment of disorders andwhere the pharmaceutical composition can include a compound according toany embodiment of the present invention, or any combination thereof, ina pharmaceutically acceptable carrier.

According to another embodiment of the invention, a process for making apharmaceutical composition is provided, the process involving combininga compound according to any embodiment of the present invention, or anycombination thereof, or its pharmaceutically acceptable salts, hydrates,solvates, crystal forms salts and individual diastereomers thereof, anda pharmaceutically acceptable carrier.

In another embodiment, a method for inhibiting growth of a tumor cell isprovided, which involves administering to a subject in need thereof aneffective amount of a compound according to any embodiment of thepresent invention, or any combination thereof, and a pharmaceuticallyacceptable carrier.

According to another embodiment, a method for treating a disordermodulated by the Wnt signaling pathway is provided, which includesadministering to a subject in need thereof an effective amount of acompound according to any embodiment of the present invention, or anycombination thereof, and a pharmaceutically acceptable carrier.

Further provided is a method for inducing apoptosis in a tumor cell,which includes administering to a subject in need thereof an effectiveamount of a compound according to any embodiment of the presentinvention, or any combination thereof, and a pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation showing dose-response behavior ofthe Wnt reporter (7xTCF-luciferase (Firefly) (pSUPERTOPFLASH)) andcontrol reporter (Renilla luciferase (pRL-SV40)) with candidate Wntantagonist A6;

FIG. 2 is a graphical representation showing Wnt responsive cell lines.

FIG. 3 is a pictoral and graphical represtnation showing a Wnt pathwayagonist.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery that signal transductionpathways regulated by Wnt can be inhibited, at least in part, byaromatic compounds. As set out in more detail below, groups of aromaticcompounds can inhibit proliferation of normal cells or tumor cellshaving Wnt modulated activity.

While not wishing to be bound by any particular theory, the activationof a receptor may be the mechanism by which these compounds act. Forexample, the compounds could affect the activity of a Wnt frizzledreceptor. Alternatively, the compounds could affect the activity of theserine/threonine kinase GSK3β, which is involved in the down regulationof β-catenin. The compounds could also affect the activity of the APCgene. In the absence of Wnt signal, the APC protein functions to fosterdegradation of β-catenin and prevent its nuclear entry. Wnt stimulation,loss of APC protein function, or of its associated partner Axin, alllead to stabilization of and concentration in the nucleus of β-catenin,which then can act as a transcriptional co-activator by associating withthe Tcf/LEF family of transcription factors. APC in complex with Axinand other proteins target β-catenin for proteasomal degradation byscaffolding the association between β-catenin and kinases whose actionlead to β-catenin ubiquitinylation; this action is abrogated byrecruitment of the degradation complex to the membrane upon Wntactivation of a receptor complex that includes Frizzled (Fz), a relativeof Smo, and LRP5/6. The pathway can also be activated by mutations ofβ-catenin that render it resistant to degradation.

Or, for example, the compounds could alter the activity of disheveled,which is a positive mediator of Wnt signaling. For example, the abilityof these compounds to inhibit proliferation of cells may be due to theability of such molecules to interact with Wnt, or at least to interferewith the ability of those proteins to activate a Wnt-mediated signaltransduction pathway. Signal transduction antagonists of differentstructures, even ones that bind to the same protein in the signalingpathways, may act in slightly different ways. Accordingly, even if aparticular condition caused or contributed to by aberrant or unwantedactivation of the Wnt pathway shows little response to treatment by oneof the antagonists disclosed herein, another of the antagonistsdisclosed herein may nonetheless be efficacious.

It is therefore specifically contemplated that these aromatic compoundsthat interfere with aspects of Wnt signal transduction activity willlikewise be capable of inhibiting proliferation (or other biologicalconsequences) in normal cells and/or cells having mutations that resultin aberrant cell proliferation. Such mutations can include, for example,mutations in the β-catenin gene or the APC gene. Thus, it iscontemplated that in certain embodiments, these compounds may be usefulfor inhibiting Wnt activity in normal cells, e.g., which do not have agenetic mutation that activates the Wnt pathway. In other embodiments,the subject inhibitors are organic molecules having a molecular weightless than 2500 amu, more preferably less than 1500 amu, and even morepreferably less than 750 amu, and are capable of inhibiting at leastsome of the biological activities of Wnt proteins, preferablyspecifically in target cells.

Thus, the methods of the present invention include the use of compounds,such as aromatic compounds, which antagonize activity of the Wnt pathwayresulting in the regulation of repair and/or functional performance of awide range of cells, tissues, and organs. In an alternative embodiment,the present invention provides compounds, such as aromatic compounds,which agonize activity of the Wnt pathway, resulting in the regulationof repair and/or functional performance of a wide range of cells,tissues, and organs. For instance, the subject methods have therapeuticand cosmetic applications ranging from regulation of neural tissues,bone and cartilage formation and repair, regulation of spermatogenesis,regulation of smooth muscle, regulation of lung, liver and other organsarising from the primative gut, regulation of hematopoietic function,regulation of skin and hair growth, etc. Moreover, the subject methodscan be performed on cells which are provided in culture (in vitro), oron cells in a whole animal (in vivo). See, for example, PCT publicationsWO 95/18856 and WO 96/17924 (the specifications of which are expresslyincorporated by reference herein).

In another embodiment, the subject method can be used to treatepithelial cells employing a compound, such as an aromatic compound,which antagonizes Wnt function, e.g., by agonizing Wnt activity. Forinstance, the subject method can be used in treating or preventing basalcell carcinoma, colon cancer, or other Wnt pathway-related disorders. Inan alternative embodiment, the subject method can be to treat epithelialcells employing an agent which agonizes hedgehog function, e.g., byantagonizing Wnt activity.

In another embodiment, the subject method can be used as part of atreatment regimen for cancer. Such caners include malignantmedulloblastoma and other primary CNS malignant neuroectodermal tumors,rhabdomyosarcoma, lung cancer, and in particular small cell lung cancer,gut-derived tumors, including but not limited to cancer of theesophagus, stomach, pancreas, and biliary duct system; prostate andbladder cancers, colon cancer, and liver cancer.

In another aspect, the present invention provides pharmaceuticalpreparations comprising, an aromatic compound such as described herein,formulated in an amount sufficient to regulate, in vivo, Wnt pathway,e.g., proliferation or other biological consequences of mis-expressionof Wnt.

The subject treatments using the subject compounds can be effective forboth human and animal subjects. Animal subjects to which the inventionis applicable extend to both domestic animals and livestock, raisedeither as pets or for commercial purposes. Examples are dogs, cats,cattle, horses, sheep, hogs, and goats.

Accordingly, the present invention is directed to heterocycliccompounds, such as heterocyclic compounds derived from benzotriazine,triazines, or pteridine, and to use of the heterocyclic compounds fortherapeutic purposes.

The following terminology and definitions apply as used in the presentapplication. The chemical terms used herein are generally in conformitywith the terminology recommended by the International Union of Pure andApplied Chemistry (IUPAC).

The phrase “aberrant modification or mutation” of a gene refers to suchgenetic lesions as, for example, deletions, substitution or addition ofnucleotides to a gene, as well as gross chromosomal rearrangements ofthe gene and/or abnormal methylation of the gene. Likewise,“mis-expression” of a gene refers to aberrant levels of transcription ofthe gene relative to those levels in a normal cell under similarconditions, as well as non-wild-type splicing of mRNA transcribed fromthe gene.

“Basal cell carcinomas” exist in a variety of clinical and histologicalforms such as nodular-ulcerative, superficial, pigmented, morphealike,fibroepithelioma and nevoid syndrome. Basal cell carcinomas are the mostcommon cutaneous neoplasms found in humans. The majority of new cases ofnonmelanoma skin cancers fall into this category.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate surrounding tissues and to giverise to metastases. Exemplary carcinomas include: “basal cellcarcinoma”, which is an epithelial tumor of the skin that, while seldommetastasizing, has potentialities for local invasion and destruction;“squamous cell carcinoma”, which refers to carcinomas arising fromsquamous epithelium and having cuboid cells; “carcinosarcoma”, whichinclude malignant tumors composed of carcinomatous and sarcomatoustissues; “adenocystic carcinoma”, carcinoma marked by cylinders or bandsof hyaline or mucinous stroma separated or surrounded by nests or cordsof small epithelial cells, occurring in the mammary and salivary glands,and mucous glands of the respiratory tract; “epidermoid carcinoma”,which refers to cancerous cells which tend to differentiate in the sameway as those of the epidermis; i.e., they tend to form prickle cells andundergo cornification; “nasopharyngeal carcinoma”, which refers to amalignant tumor arising in the epithelial lining of the space behind thenose; and “renal cell carcinoma”, which pertains to carcinoma of therenal parenchyma composed of tubular cells in varying arrangements.Other carcinomatous epithelial growths are “papillomas”, which refers tobenign tumors derived from epithelium and having a papillomavirus as acausative agent; and “epidermoidomas”, which refers to a cerebral ormeningeal tumor formed by inclusion of ectodermal elements at the timeof closure of the neural groove.

The “corium” or “dermis” refers to the layer of the skin deep to theepidermis, consisting of a dense bed of vascular connective tissue, andcontaining the nerves and terminal organs of sensation. The hair roots,and sebaceous and sweat glands are structures of the epidermis which aredeeply embedded in the dermis.

“Dental tissue” refers to tissue in the mouth which is similar toepithelial tissue, for example gum tissue. The method of the presentinvention is useful for treating periodontal disease.

“Dermal skin ulcers” refer to lesions on the skin caused by superficialloss of tissue, usually with inflammation. Dermal skin ulcers which canbe treated by the method of the present invention include decubitusulcers, diabetic ulcers, venous stasis ulcers and arterial ulcers.Decubitus wounds refer to chronic ulcers that result from pressureapplied to areas of the skin for extended periods of time. Wounds ofthis type are often called bedsores or pressure sores. Venous stasisulcers result from the stagnation of blood or other fluids fromdefective veins. Arterial ulcers refer to necrotic skin in the areaaround arteries having poor blood flow.

The term “ED₅₀” means the dose of a drug which produces 50% of itsmaximum response or effect.

The terms “epithelia”, “epithelial” and “epithelium” refer to thecellular covering of internal and external body surfaces (cutaneous,mucous and serous), including the glands and other structures derivedtherefrom, e.g., corneal, esophegeal, epidermal, and hair follicleepithelial cells. Other exemplary epithlelial tissue includes: olfactoryepithelium, which is the pseudostratified epithelium lining theolfactory region of the nasal cavity, and containing the receptors forthe sense of smell; glandular epithelium, which refers to epitheliumcomposed of secreting cells; squamous epithelium, which refers toepithelium composed of flattened plate-like cells. The term epitheliumcan also refer to transitional epithelium, like that which ischaracteristically found lining hollow organs that are subject to greatmechanical change due to contraction and distention, e.g., tissue whichrepresents a transition between stratified squamous and columnarepithelium.

The term “epithelialization” refers to healing by the growth ofepithelial tissue over a denuded surface.

The term “epidermal gland” refers to an aggregation of cells associatedwith the epidermis and specialized to secrete or excrete materials notrelated to their ordinary metabolic needs. For example, “sebaceousglands” are holocrine glands in the corium that secrete an oilysubstance and sebum. The term “sweat glands” refers to glands thatsecrete sweat, situated in the corium or subcutaneous tissue, opening bya duct on the body surface.

The term “epidermis” refers to the outermost and nonvascular layer ofthe skin, derived from the embryonic ectoderm, varying in thickness from0.07-1.4 mm. On the palmar and plantar surfaces it comprises, fromwithin outward, five layers: basal layer composed of columnar cellsarranged perpendicularly; prickle-cell or spinous layer composed offlattened polyhedral cells with short processes or spines; granularlayer composed of flattened granular cells; clear layer composed ofseveral layers of clear, transparent cells in which the nuclei areindistinct or absent; and horny layer composed of flattened, cornifiednon-nucleated cells. In the epidermis of the general body surface, theclear layer is usually absent.

The “growth state” of a cell refers to the rate of proliferation of thecell and/or the state of differentiation of the cell. An “altered growthstate” is a growth state characterized by an abnormal rate ofproliferation, e.g., a cell exhibiting an increased or decreased rate ofproliferation relative to a normal cell.

The term “agonist” refers to an agent or analog that binds productivelyto a receptor and mimics its biological activity. The term “antagonist”refers to an agent that binds to receptors but does not provoke thenormal biological response. Thus, an antagonist potentiates orrecapitulates, for example, the bioactivity of patched, such as torepress transcription of target genes. The term “Wnt antagonist” as usedherein refers not only to any agent that may act by directly inhibitingthe normal function of the Wnt protein, but also to any agent thatinhibits the Wnt signaling pathway, and thus recapitulates the functionof Wnt. The term “Wnt agonist” likewise refers to an agent whichantagonizes or blocks the bioactivity of Wnt, such as to increasetranscription of target genes.

As used herein, “immortalized cells” refers to cells which have beenaltered via chemical and/or recombinant means such that the cells havethe ability to grow through an indefinite number of divisions inculture.

“Internal epithelial tissue” refers to tissue inside the body which hascharacteristics similar to the epidermal layer in the skin. Examplesinclude the lining of the intestine. The method of the present inventionis useful for promoting the healing of certain internal wounds, forexample wounds resulting from surgery.

The term “keratosis” refers to proliferative skin disorder characterizedby hyperplasia of the horny layer of the epidermis. Exemplary keratoticdisorders include keratosis follicularis, keratosis palmaris etplantaris, keratosis pharyngea, keratosis pilaris, and actinickeratosis.

The term “LD₅₀” means the dose of a drug which is lethal in 50% of testsubjects.

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human animal.

The term “prodrug” is intended to encompass compounds which, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties which are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity of the host animal.

As used herein, “proliferating” and “proliferation” refer to cellsundergoing mitosis.

The term “proliferative skin disorder” refers to any disease/disorder ofthe skin marked by unwanted or aberrant proliferation of cutaneoustissue. These conditions are typically characterized by epidermal cellproliferation or incomplete cell differentiation, and include, forexample, X-linked ichthyosis, psoriasis, atopic dermatitis, allergiccontact dermatitis, epidermolytic hyperkeratosis, and seborrheicdermatitis. For example, epidermodysplasia is a form of faultydevelopment of the epidermis. Another example is “epidermolysis”, whichrefers to a loosened state of the epidermis with formation of blebs andbullae either spontaneously or at the site of trauma.

The term “psoriasis” refers to a hyperproliferative skin disorder whichalters the skin's regulatory mechanisms. In particular, lesions areformed which involve primary and secondary alterations in epidermalproliferation, inflammatory responses of the skin, and an expression ofregulatory molecules such as lymphokines and inflammatory factors.Psoriatic skin is morphologically characterized by an increased turnoverof epidermal cells, thickened epidermis, abnormal keratinization,inflammatory cell infiltrates into the dermis layer andpolymorphonuclear leukocyte infiltration into the epidermis layerresulting in an increase in the basal cell cycle. Additionally,hyperkeratotic and parakeratotic cells are present.

The term “transformed cells” refers to cells which have spontaneouslyconverted to a state of unrestrained growth, i.e., they have acquiredthe ability to grow through an indefinite number of divisions inculture. Transformed cells may be characterized by such terms asneoplastic, anaplastic and/or hyperplastic, with respect to their lossof growth control.

The term “heterocyclic,” when used to describe an aromatic ring, meansthat the aromatic ring contains at least one heteroatom. Theabbreviation “Het” is sometimes used to signify a heterocyclicstructure.

The term “heteroatom” is defined to include any atom other than carbon,such as nitrogen.

The term “aromatic” or “aryl” is defined to include a cyclicallyconjugated molecular entity with a stability, due to delocalization,significantly greater than that of a hypothetical localized structure,such as the Kekulé structure.

The term “heterocyclic,” when not used to describe an aromatic ring, isdefined to include cyclic (i.e., ring-containing) groups other thanaromatic groups, the cyclic group being formed by between 3 and about 14carbon atoms and at least one heteroatom described above. The term“substituted heterocyclic” is defined to include both aromatic andnon-aromatic structures to heterocyclic groups further bearing one ormore substituents.

The term “heteroaryl” is defined to include aromatic rings, where thering structure is formed by between 3 and about 14 carbon atoms and byat least one heteroatom described above, and the term “substitutedheteroaryl” refers to heteroaryl groups further bearing one or moresubstituents.

The term “fused” or “fused rings” is defined as polycyclic ring systemin which any two adjacent rings have at least two adjacent atoms incommon.

The term “alkyl” is defined to include a monovalent straight or branchedchain hydrocarbon group having from one to about 12 carbon atoms, forexample, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,tert-butyl, n-pentyl (also known as n-amyl), n-hexyl, and the like.

The term “alkylaryl” is defined to include an alkyl group substitutedwith an aryl group, such as phenyl group.

The term “halogen” is defined to include an atom of fluorine, chlorine,bromine or iodine.

The term “N-oxide” is defined to include nitrogen-containingheterocyclic moieties in which at least one nitrogen atom is associatedwith oxygen to form the structure N→O.

The term “phenyl” is defined to include moieties having structure (Ph):

The term “benzyl” is defined to include moieties having structure (Bz):

The term “naphthalene-1,4-dione” is defined to include moieties havingstructure (NPD):

The term “triazole” is defined to include moieties having structure(TA):

The term “triazole-N-oxide” is defined to include moieties havingstructure (TANO):

The term “piperazine” is defined to include moieties having structure(PP):

The term “piperazinyl” is defined to include moieties having structure(PP1):

The term “benzopiperazine ” is defined to include moieties havingstructure (BPP):

The term “imidazoe” is defined to include moieties having structure(IAZ):

The term “azaanthracene” is defined to include moieties having structure(AA):

The term “piperidyl” is defined to include moieties having structure(PP):

The term “effective amount” is defined as the amount of the compound orpharmaceutical composition that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought bythe researcher, veterinarian, medical doctor or other clinician, e.g.,restoration or maintenance of vasculostasis or prevention of thecompromise or loss or vasculostasis; reduction of tumor burden;reduction of morbidity and/or mortality. For example, a “therapeuticallyeffective amount” of, e.g., an aromatic compound, with respect to thesubject method of treatment, refers to an amount of the Wnt antagonistin a preparation which, when applied as part of a desired dosage regimenbrings about, e.g., a change in the rate of cell proliferation and/orthe state of differentiation of a cell and/or rate of survival of a cellaccording to clinically acceptable standards for the disorder to betreated or the cosmetic purpose.

The term “pharmaceutically acceptable” is defined as a carrier, whetherdiluent or excipient, that is compatible with the other ingredients ofthe formulation and not deleterious to the recipient thereof.

The terms “administration of a compound” or “administering a compound”is defined to include an act of providing a compound of the invention orpharmaceutical composition to the subject in need of treatment.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit signaltransduction pathways), wherein one or more simple variations ofsubstituents are made which do not adversely affect the efficacy of thecompound. In general, the compounds of the present invention may beprepared by the methods known in the art, or by modifications thereof,using readily available starting materials, reagents and conventionalsynthesis procedures. In these reactions, it is also possible to makeuse of variants which are in themselves known, but are not mentionedhere.

As described in further detail below, it is contemplated that thesubject methods can be carried out using a variety of different aromaticcompounds, which can be readily identified, e.g., by such drug screeningassays as described herein.

According to an embodiment of the invention, a first type of compoundsis provided for treatment of various diseases, disorders, andpathologies. The compounds of the first type can include a firstaromatic moiety fused to a second aromatic moiety, wherein the firstaromatic moiety can be naphthalene-1,4-dione moiety and the secondaromatic moiety can be an N-substituted triazole-N-oxide moiety. Thesubstitutent in the N-substituted triazole-N-oxide moiety can include analkylaryl group. One example of the alkylaryl group that can be usedincludes benzyl group.

An example of a compound of the first type, that can be used, is thecompound having formula (I), or a pharmaceutically acceptable saltthereof:

According to an embodiment of the invention, a second type of compoundsis provided for treatment of various diseases, disorders, andpathologies. The compounds of the second type can include abenzopiperazine moiety fused to a substituted imidazole moiety. Anexample of the benzopiperazine moiety that can be used includes analkylpiperazinyl group, such as methylpirazinyl group. An exemplaryimidazole moiety that can be used includes a phenyl substitutent. Inaddition to the phenyl substitutent, the imidazole moiety can furtherinclude a halogenated aromatic group attached to a nitrogen atom in theimidazole structure, e.g., a bromophenyl group.

An example of a compound of the second type that can be used is thecompound having the formula (II), or a pharmaceutically acceptable saltthereof:

According to an embodiment of the invention, a third type of compoundsis provided for treatment of various diseases, disorders, andpathologies. The compounds of the third type can include anazaanthracene moiety and a secondary amino moiety, or a pharmaceuticallyacceptable salt thereof. For example, the secondary amino moiety can beattached to the nitrogen-containing ring of the azaanthracene moiety. Anexemplary secondary amino moiety can include a piperidyl group. Thenitrogen of the piperidyl group can be further substituted with an alkylgroup, e.g., with ethyl group.

An example of a compound of the third type that can be used is thecompound having the formula (III), or a pharmaceutically acceptable saltthereof:

The compounds described above can be prepared by methods known in theart.

In certain embodiments, the subject aromatic compounds can be chosen onthe basis of their selectively for the Wnt pathway. This selectivity canbe for the Wnt pathway versus other steroid-mediated pathways (such astestosterone or estrogen mediated activities), as well as selectivityfor particular Wnt pathways, e.g., which isotype specific for Wnt. Forinstance, the subject method may employ aromatic compounds that do notsubstantially interfere with the biological activity of such steroids asaldosterone, androstane, androstene, androstenedione, androsterone,cholecalciferol, cholestane, cholic acid, corticosterone, cortisol,cortisol acetate, cortisone, cortisone acetate, deoxycorticosterone,digitoxigenin, ergocalciferol, ergosterol, estradiol-17-α,estradiol-17-β, estriol, estrane, estrone, hydrocortisone, lanosterol,lithocholic acid, mestranol, β-methasone, prednisone, pregnane,pregnenolone, progesterone, spironolactone, testosterone, triamcinoloneand their derivatives, at least so far as those activities are unrelatedto Wnt-related signaling.

In one embodiment, the subject aromatic compounds for use in the presentmethods have a k_(d) for members of the nuclear hormone receptorsuperfamily of greater than 1 μM, and more preferably greater than 1 mM,e.g., it does not bind estrogen, testosterone receptors or the like. Inanother embodiment, the subject agonist has no estrogenic activity atphysiological concentrations (e.g., in the range of 1 ng-1 mg/kg).

Thus, in one embodiment, untoward side effects that may be associatedwith certain members of the aromatic compounds can be reduced by, forexample, using the drug screening assays described herein. Theapplication of combinatorial and medicinal chemistry techniques to thearomatic compounds provides a means for reducing such unwanted negativeside effects including personality changes, shortened life spans,cardiovascular diseases and vascular occlusion., organ toxicity,hyperglycemia and diabetes, Cushnoid features, “wasting” syndrome,steroidal glaucoma, hypertension, peptic ulcers, and increasedsusceptibility to infections. For certain embodiments, it will bebeneficial to reduce the teratogenic activity relative to jervine, asfor example, in the use of the subject method to selectively inhibitspermatogenesis.

Another aspect of the present invention relates to a method ofmodulating a differentiated state, survival, and/or proliferation of acell having Wnt receptor, by contacting the cells with an aromaticcompound as set forth above according to the subject method and as thecircumstances may warrant.

It is contemplated by the invention that, in light of the findings of anapparently broad involvement of Wnt in the formation of ordered spatialarrangements of differentiated tissues in vertebrates, the subjectmethod could be used as part of a process for generating and/ormaintaining an array of different vertebrate tissue both in vitro and invivo. The aromatic compound, whether inductive or anti-inductive withrespect proliferation or differentiation of a given tissue, can be, asappropriate, any of the preparations described above.

For example, the present method of using the subject aromatic compoundsis applicable to cell culture techniques wherein, whether for genetic orbiochemical reasons, the cells have a Wnt receptor. Alternatively, asubject aromatic compound may be employed in a related method directedtowards cells which have a Wnt receptor. In vitro neuronal culturesystems have proven to be fundamental and indispensable tools for thestudy of neural development, as well as the identification ofneurotrophic factors such as nerve growth factor (NGF), ciliary trophicfactors (CNTF), and brain derived neurotrophic factor (BDNF). One use ofthe present method may be in cultures of neuronal stem cells, such as inthe use of such cultures for the generation of new neurons and glia. Insuch embodiments of the subject method, the cultured cells can becontacted with an aromatic compound of the present invention in order toalter the rate of proliferation of neuronal stem cells in the cultureand/or alter the rate of differentiation, or to maintain the integrityof a culture of certain terminally differentiated neuronal cells. In anexemplary embodiment, the subject method can be used to culture, forexample, sensory neurons or, alternatively, motorneurons: Such neuronalcultures can be used as convenient assay systems as well as sources ofimplantable cells for therapeutic treatments.

In another embodiment, the subject method can be used in the treatmentof neoplastic or hyperplastic transformations such as may occur in thecentral nervous system. For instance, the subject compounds can beutilized to cause such transformed cells to become either post-mitoticor apoptotic. The present method may, therefore, be used as part of atreatment for, e.g., malignant gliomas, meningiomas, medulloblastomas,neuroectodermal tumors, and ependymomas.

In another embodiment, the subject method can be used as part of atreatment regimen for malignant medulloblastoma and other primary CNSmalignant neuroectodermal tumors.

In certain embodiments, the subject method is used as part of treatmentprogram for medulloblastoma. Medulloblastoma, a primary brain tumor, isthe most common brain tumor in children. A medulloblastoma is aprimitive neuroectodermal tumor arising in the posterior fossa. Theyaccount for approximately 25% of all pediatric brain tumors (Miller).Histologically, they are small round cell tumors commonly arranged intrue rosettes, but may display some differentiation to astrocytes,ependymal cells or neurons (Rorke; Kleihues). PNET's may arise in otherareas of the brain including the pineal gland (pineoblastoma) andcerebrum. Those arising in the supratentorial region generally fareworse than their PF counterparts.

Medulloblastoma/PNET's are known to recur anywhere in the CNS afterresection, and can even metastasize to bone. Pretreatment evaluationshould therefore include an examination of the spinal cord to excludethe possibility of “dropped metastases”. Gadolinium-enhanced MRI haslargely replaced myelography for this purpose, and CSF cytology isobtained postoperatively as a routine procedure.

In other embodiments, the subject method is used as part of a treatmentprogram for hepatocellular carcinoma. Hepatocellular carcinoma is a formof cancer that arises from hepatocytes, the major cell type of the live,and is one of the most common tumors involving mutations in the Wntpathway.

In other embodiments, the subject method is used as part of treatmentprogram for ependymomas. Ependymomas account for approximately 10% ofthe pediatric brain tumors in children. Grossly, they are tumors thatarise from the ependymal lining of the ventricles and microscopicallyform rosettes, canals, and perivascular rosettes. In the CHOP series of51 children reported with ependymomas, ¾ were histologically benign.Approximately ⅔ arose from the region of the 4th ventricle. One thirdpresented in the supratentorial region. Age at presentation peaksbetween birth and 4 years, as demonstrated by SEER data as well as datafrom CHOP. The median age is about 5 years. Because so many childrenwith this disease are babies, they often require multimodal therapy.

Yet another aspect of the present invention concerns the observation inthe art that Wnt is involved in morphogenic signals involved in othervertebrate organogenic pathways in addition to neuronal differentiationas described above, having apparent roles in other endodermalpatterning, as well as both mesodermal and endodermal differentiationprocesses. Thus, it is contemplated by the invention that compositionscomprising one or more of the subject compounds can also be utilized forboth cell culture and therapeutic methods involving generation andmaintenance of non-neuronal tissue.

In one embodiment, the present invention makes use of the discovery thatWnt is apparently involved in controlling the development of stem cellsresponsible for formation of the digestive tract, liver, lungs, andother organs which derive from the primitive gut. Shh serves as aninductive signal from the endoderm to the mesoderm, which is critical togut morphogenesis. Therefore, for example, compounds of the instantmethod can be employed for regulating the development and maintenance ofan artificial liver which can have multiple metabolic functions of anormal liver. In an exemplary embodiment, the subject method can be usedto regulate the proliferation and differentiation of digestive tube stemcells to form hepatocyte cultures which can be used to populateextracellular matrices, or which can be encapsulated in biocompatiblepolymers, to form both implantable and extracorporeal artificial livers.

In another embodiment, therapeutic compositions of subject compounds canbe utilized in conjunction with transplantation of such artificiallivers, as well as embryonic liver structures, to regulate uptake ofintraperitoneal implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted liver tissue.

In yet another embodiment, the subject method can be employedtherapeutically to regulate such organs after physical, chemical orpathological insult. For instance, therapeutic compositions comprisingsubject compounds can be utilized in liver repair subsequent to apartial hepatectomy.

The generation of the pancreas and small intestine from the embryonicgut depends on intercellular signalling between the endodermal andmesodermal cells of the gut. In particular, the differentiation ofintestinal mesoderm into smooth muscle has been suggested to depend onsignals from adjacent endodermal cells. One candidate mediator ofendodermally derived signals in the embryonic hindgut is Sonic hedgehog.See, for example, Apelqvist et al., Curr. Biol. 7:801-4 (1997). The Shhgene is expressed throughout the embryonic gut endoderm with theexception of the pancreatic bud endoderm, which instead expresses highlevels of the homeodomain protein Ipf1/Pdx1 (insulin promoter factor1/pancreatic and duodenal homeobox 1), an essential regulator of earlypancreatic development. Apelqvist et al., supra, have examined whetherthe differential expression of Shh in the embryonic gut tube controlsthe differentiation of the surrounding mesoderm into specialisedmesoderm derivatives of the small intestine and pancreas. To test this,they used the promoter of the Ipf1/Pdx1 gene to selectively express Shhin the developing pancreatic epithelium. In Ipf1/Pdx1-Shh transgenicmice, the pancreatic mesoderm developed into smooth muscle andinterstitial cells of Cajal, characteristic of the intestine, ratherthan into pancreatic mesenchyme and spleen. Also, pancreatic explantsexposed to Shh underwent a similar program of intestinaldifferentiation. These results provide evidence that the differentialexpression of endodermally derived Shh controls the fate of adjacentmesoderm at different regions of the gut tube.

In the context of the present invention, it is contemplated thereforethat the subject compounds can be used to control or regulate theproliferation and/or differentiation of pancreatic tissue both in vivoand in vitro.

There are a wide variety of pathological cell proliferative anddifferentiative conditions for which the aromatic compounds of thepresent invention may provide therapeutic benefits, with the generalstrategy being, for example, the correction of aberrant insulinexpression, or modulation of differentiation. More generally, however,the present invention relates to a method of inducing and/or maintaininga differentiated state, enhancing survival and/or affectingproliferation of pancreatic cells, by contacting the cells with thesubject inhibitors. For instance, it is contemplated by the inventionthat, in light of the apparent involvement of Wnt in the formation ofordered spatial arrangements of pancreatic tissues, the subject methodcould be used as part of a technique to generate and/or maintain suchtissue both in vitro and in vivo. For instance, modulation of thefunction of Wnt can be employed in both cell culture and therapeuticmethods involving generation and maintenance β-cells and possibly alsofor non-pancreatic tissue, such as in controlling the development andmaintenance of tissue from the digestive tract, spleen, lungs, colon,and other organs which derive from the primitive gut.

In an exemplary embodiment, the present method can be used in thetreatment of hyperplastic and neoplastic disorders effecting pancreatictissue, particularly those characterized by aberrant proliferation ofpancreatic cells. For instance, pancreatic cancers are marked byabnormal proliferation of pancreatic cells which can result inalterations of insulin secretory capacity of the pancreas. For instance,certain pancreatic hyperplasias, such as pancreatic carcinomas, canresult in hypoinsulinemia due to dysfunction of β-cells or decreasedislet cell mass. To the extent that aberrant Wnt signaling may beindicated in disease progression, the subject aromatic compounds can beused to enhance regeneration of the tissue after anti-tumor therapy.

Moreover, manipulation of Wnt signaling properties at different pointsmay be useful as part of a strategy for reshaping/repairing pancreatictissue both in vivo and in vitro. In one embodiment, the presentinvention makes use of the apparent involvement of Wnt in regulating thedevelopment of pancreatic tissue. In general, the subject method can beemployed therapeutically to regulate the pancreas after physical,chemical or pathological insult. In yet another embodiment, the subjectmethod can be applied to cell culture techniques, and in particular, maybe employed to enhance the initial generation of prosthetic pancreatictissue devices. Manipulation of proliferation and differentiation ofpancreatic tissue, for example, by altering Wnt, can provide a means formore carefully controlling the characteristics of a cultured tissue. Inan exemplary embodiment, the subject method can be used to augmentproduction of prosthetic devices which require β-islet cells, such asmay be used in the encapsulation devices described in, for example, theAebischer et al. U.S. Pat. No. 4,892,538, the Aebischer et al. U.S. Pat.No. 5,106,627, the Lim U.S. Pat. No. 4,391,909, and the Sefton U.S. Pat.No. 4,353,888. Early progenitor cells to the pancreatic islets aremultipotential, and apparently coactivate all the islet-specific genesfrom the time they first appear. As development proceeds, expression ofislet-specific hormones, such as insulin, becomes restricted to thepattern of expression characteristic of mature islet cells. Thephenotype of mature islet cells, however, is not stable in culture, asreappearance of embryonal traits in mature β-cells can be observed. Byutilizing the subject compounds, the differentiation path orproliferative index of the cells can be regulated.

Furthermore, manipulation of the differentiative state of pancreatictissue can be utilized in conjunction with transplantation of artificialpancreas so as to promote implantation, vascularization, and in vivodifferentiation and maintenance of the engrafted tissue. For instance,manipulation of Wnt function to affect tissue differentiation can beutilized as a means of maintaining graft viability.

Bellusci et al., Development 124:53 (1997) report that Sonic hedgehogregulates lung mesenchymal cell proliferation in vivo. Accordingly, thepresent method can be used to regulate regeneration of lung tissue,e.g., in the treatment of emphysema.

Fujita et al., Biochem. Biophys. Res. Commun. 238:658 (1997) reportedthat Sonic hedgehog is expressed in human lung squamous carcinoma andadenocarcinoma cells. The expression of Sonic hedgehog was also detectedin the human lung squamous carcinoma tissues, but not in the normal lungtissue of the same patient. They also observed that Sonic hedgehogstimulates the incorporation of BrdU into the carcinoma cells andstimulates their cell growth, while anti-Shh-N inhibited their cellgrowth. These results suggest that ptc and/or Wnt is involved in thecell growth of such transformed lung tissue and therefore indicates thatthe subject method can be used as part of a treatment of lung carcinomaand adenocarcinomas, and other proliferative disorders involving thelung epithelia.

Many other tumors may, based on evidence such as involvement of the Wntpathway in these tumors, or detected expression of Wnt or its receptorsin these tissues during development, be affected by treatment with thesubject aromatic compounds. Such tumors include, but are by no meanslimited to, tumors related to Gorlin's syndrome (e.g., basal cellcarcinoma, medulloblastoma, meningioma, etc.), tumors evidenced in pctknock-out mice (e.g., hemangioma, rhabdomyosarcoma, etc.), tumorsresulting from gli-1 amplification (e.g., glioblastoma, sarcoma, etc.),tumors connected with TRC8, a ptc homolog (e.g., renal carcinoma,thyroid carcinoma, etc.), Ext-1-related tumors (e.g., bone cancer,etc.), Shh-induced tumors (e.g., lung cancer, chondrosarcomas, etc.),and other tumors (e.g., breast cancer, urogenital cancer (e.g., kidney,bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinalcancer (e.g., stomach, intestine, etc.), etc.).

In still another embodiment of the present invention, compositionscomprising one or more of the subject compounds can be used in the invitro generation of skeletal tissue, such as from skeletogenic stemcells, as well as the in vivo treatment of skeletal tissue deficiencies.The present invention particularly contemplates the use of subjectcompounds to regulate the rate of chondrogenesis and/or osteogenesis. By“skeletal tissue deficiency”, it is meant a deficiency in bone or otherskeletal connective tissue at any site where it is desired to restorethe bone or connective tissue, no matter how the deficiency originated,e.g. whether as a result of surgical intervention, removal of tumor,ulceration, implant, fracture, or other traumatic or degenerativeconditions.

For instance, the methods of the present invention can be used as partof a regimen for restoring cartilage function to a connective tissue.Such methods are useful in, for example, the repair of defects orlesions in cartilage tissue which is the result of degenerative wearsuch as that which results in arthritis, as well as other mechanicalderangements which may be caused by trauma to the tissue, such as adisplacement of torn meniscus tissue, meniscectomy, a laxation of ajoint by a torn ligament, malignment of joints, bone fracture, or byhereditary disease. The present reparative method is also useful forremodeling cartilage matrix, such as in plastic or reconstructivesurgery, as well as periodontal surgery. The present method may also beapplied to improving a previous reparative procedure, for example,following surgical repair of a meniscus, ligament, or cartilage.Furthermore, it may prevent the onset or exacerbation of degenerativedisease if applied early enough after trauma.

In one embodiment of the present invention, the subject method comprisestreating the afflicted connective tissue with a therapeuticallyeffective amount of a subject aromatic compound to regulate a cartilagerepair response in the connective tissue by managing the rate ofdifferentiation and/or proliferation of chondrocytes embedded in thetissue. Such connective tissues as articular cartilage, interarticularcartilage (menisci), costal cartilage (connecting the true ribs and thesternum), ligaments, and tendons are particularly amenable to treatmentin reconstructive and/or regenerative therapies using the subjectmethod. As used herein, regenerative therapies include treatment ofdegenerative states which have progressed to the point of whichimpairment of the tissue is obviously manifest, as well as preventivetreatments of tissue where degeneration is in its earliest stages orimminent.

In an illustrative embodiment, the subject method can be used as part ofa therapeutic intervention in the treatment of cartilage of adiarthroidal joint, such as a knee, an ankle, an elbow, a hip, a wrist,a knuckle of either a finger or toe, or a tempomandibular joint. Thetreatment can be directed to the meniscus of the joint, to the articularcartilage of the joint, or both. To further illustrate, the subjectmethod can be used to treat a degenerative disorder of a knee, such aswhich might be the result of traumatic injury (e.g., a sports injury orexcessive wear) or osteoarthritis. The subject regulators may beadministered as an injection into the joint with, for instance, anarthroscopic needle. In some instances, the injected agent can be in theform of a hydrogel or other slow release vehicle described above inorder to permit a more extended and regular contact of the agent withthe treated tissue.

The present invention further contemplates the use of the subjectmethods in the field of cartilage transplantation and prosthetic devicetherapies. However, problems arise, for instance, because thecharacteristics of cartilage and fibrocartilage varies between differenttissue: such as between articular, meniscal cartilage, ligaments, andtendons, between the two ends of the same ligament or tendon, andbetween the superficial and deep parts of the tissue. The zonalarrangement of these tissues may reflect a gradual change in mechanicalproperties, and failure occurs when implanted tissue, which has notdifferentiated under those conditions, lacks the ability toappropriately respond. For instance, when meniscal cartilage is used torepair anterior cruciate ligaments, the tissue undergoes a metaplasia topure fibrous tissue. By regulating the rate of chondrogenesis, thesubject method can be used to particularly address this problem, byhelping to adaptively control the implanted cells in the new environmentand effectively resemble hypertrophic chondrocytes of an earlierdevelopmental stage of the tissue.

In similar fashion, the subject method can be applied to enhancing boththe generation of prosthetic cartilage devices and to theirimplantation. The need for improved treatment has motivated researchaimed at creating new cartilage that is based oncollagen-glycosaminoglycan templates (Stone et al., Clin. Orthop. Relat.Red 252:129 (1990)), isolated chondrocytes (Grande et al., J. Orthop.Res. 7:208 (1989); and Takigawa et al., Bone Miner 2:449 (1987)), andchondrocytes attached to natural or synthetic polymers (Walitani et al.,J. Bone Jt. Surg. 71B:74 (1989); Vacanti et al., Plast. Reconstr. Surg.88:753 (1991); von Schroeder et al. J. Biomed. Mater. Res. 25:329(1991); Freed et al., J. Biomed. Mater. Res. 27:11 (1993); and theVacanti et al. U.S. Pat. No.5,041,138). For example, chondrocytes can begrown in culture on biodegradable, biocompatible highly porous scaffoldsformed from polymers such as polyglycolic acid, polylactic acid, agarosegel, or other polymers which degrade over time as function of hydrolysisof the polymer backbone into innocuous monomers. The matrices aredesigned to allow adequate nutrient and gas exchange to the cells untilengraftment occurs. The cells can be cultured in vitro until adequatecell volume and density has developed for the cells to be implanted. Oneadvantage of the matrices is that they can be cast or molded into adesired shape on an individual basis, so that the final product closelyresembles the patient's own ear or nose (by way of example), or flexiblematrices can be used which allow for manipulation at the time ofimplantation, as in a joint.

In one embodiment of the subject method, the implants are contacted witha subject aromatic compound during certain stages of the culturingprocess in order to manage the rate of differentiation of chondrocytesand the formation of hypertrophic chrondrocytes in the culture.

In another embodiment, the implanted device is treated with a subjectaromatic compound in order to actively remodel the implanted matrix andto make it more suitable for its intended function. As set out abovewith respect to tissue transplants, the artificial transplants sufferfrom the same deficiency of not being derived in a setting which iscomparable to the actual mechanical environment in which the matrix isimplanted. The ability to regulate the chondrocytes in the matrix by thesubject method can allow the implant to acquire characteristics similarto the tissue for which it is intended to replace.

In yet another embodiment, the subject method is used to enhanceattachment of prosthetic devices. To illustrate, the subject method canbe used in the implantation of a periodontal prosthesis, wherein thetreatment of the surrounding connective tissue stimulates formation ofperiodontal ligament about the prosthesis.

In other embodiments, the subject methods can be employed as part of aregimen for the generation of bone (osteogenesis) at a site in theanimal where such skeletal tissue is deficient. Indian hedgehog (Ihh) isparticularly associated with the hypertrophic chondrocytes that areultimately replaced by osteoblasts. For instance, administration of acompound of the present invention can be employed as part of a methodfor regulating the rate of bone loss in a subject. For example,preparations comprising subject compounds can be employed, for example,to control endochondral ossification in the formation of a “model” forossification.

The subject method also has wide applicability to the treatment orprophylaxis of disorders afflicting epithelial tissue, as well as incosmetic uses. In general, the method can be characterized as includinga step of administering to an animal an amount of a subject aromaticcompound effective to alter the growth state of a treated epithelialtissue. The mode of administration and dosage regimens will varydepending on the epithelial tissue(s) which is to be treated. Forexample, topical formulations will be preferred where the treated tissueis epidermal tissue, such as dermal or mucosal tissues.

A method which “promotes the healing of a wound” results in the woundhealing more quickly as a result of the treatment than a similar woundheals in the absence of the treatment. “Promotion of wound healing” canalso mean that the method regulates the proliferation and/or growth of,inter alia, keratinocytes, or that the wound heals with less scarring,less wound contraction, less collagen deposition and more superficialsurface area. In certain instances, “promotion of wound healing” canalso mean that certain methods of wound healing have improved successrates, (e.g., the take rates of skin grafts) when used together with themethod of the present invention.

Despite significant progress in reconstructive surgical techniques,scarring can be an important obstacle in regaining normal function andappearance of healed skin. This is particularly true when pathologicscarring such as keloids or hypertrophic scars of the hands or facecauses functional disability or physical deformity. In the severestcircumstances, such scarring may precipitate psychosocial distress and alife of economic deprivation. Wound repair includes the stages ofhemostasis, inflammation, proliferation, and remodeling. Theproliferative stage involves multiplication of fibroblasts andendothelial and epithelial cells. Through the use of the subject method,the rate of proliferation of epithelial cells in and proximal to thewound can be controlled in order to accelerate closure of the woundand/or minimize the formation of scar tissue.

The present treatment can also be effective as part of a therapeuticregimen for treating oral and paraoral ulcers, e.g., resulting fromradiation and/or chemotherapy. Such ulcers commonly develop within daysafter chemotherapy or radiation therapy. These ulcers usually begin assmall, painful irregularly shaped lesions usually covered by a delicategray necrotic membrane and surrounded by inflammatory tissue. In manyinstances, lack of treatment results in proliferation of tissue aroundthe periphery of the lesion on an inflammatory basis. For instance, theepithelium bordering the ulcer usually demonstrates proliferativeactivity, resulting in loss of continuity of surface epithelium. Theselesions, because of their size and loss of epithelial integrity, disposethe body to potential secondary infection. Routine ingestion of food andwater becomes a very painful event and, if the ulcers proliferatethroughout the alimentary canal, diarrhea usually is evident with allits complicating factors. According to the present invention, atreatment for such ulcers which includes application of a subjectcompound can reduce the abnormal proliferation and differentiation ofthe affected epithelium, helping to reduce the severity of subsequentinflammatory events.

The subject method and compositions can also be used to treat woundsresulting from dermatological diseases, such as lesions resulting fromautoimmune disorders such as psoriasis. Atopic dermititis refers to skintrauma resulting from allergies associated with an immune responsecaused by allergens such as pollens, foods, dander, insect venoms andplant toxins.

In other embodiments, antiproliferative preparations of subjectcompounds can be used to inhibit lens epithelial cell proliferation toprevent post-operative complications of extracapsular cataractextraction. Cataract is an intractable eye disease and various studieson a treatment of cataract have been made. But at present, the treatmentof cataract is attained by surgical operations. Cataract surgery hasbeen applied for a long time and various operative methods have beenexamined. Extracapsular lens extraction has become the method of choicefor removing cataracts. The major medical advantages of this techniqueover intracapsular extraction are lower incidence of aphakic cystoidmacular edema and retinal detachment. Extracapsular extraction is alsorequired for implantation of posterior chamber type intraocular lenseswhich are now considered to be the lenses of choice in most cases.

However, a disadvantage of extracapsular cataract extraction is the highincidence of posterior lens capsule opacification, often calledafter-cataract, which can occur in up to 50% of cases within three yearsafter surgery. After-cataract is caused by proliferation of equatorialand anterior capsule lens epithelial cells which remain afterextracapsular lens extraction. These cells proliferate to causeSommerling rings, and along with fibroblasts which also deposit andoccur on the posterior capsule, cause opacification of the posteriorcapsule, which interferes with vision. Prevention of after-cataractwould be preferable to treatment. To inhibit secondary cataractformation, the subject method provides a means for inhibitingproliferation of the remaining lens epithelial cells. For example, suchcells can be induced to remain quiescent by instilling a solutioncontaining a preparation of a subject compound into the anterior chamberof the eye after lens removal. Furthermore, the solution can beosmotically balanced to provide minimal effective dosage when instilledinto the anterior chamber of the eye, thereby inhibiting subcapsularepithelial growth with some specificity.

The subject methods can also be used in the treatment of corneopathiesmarked by corneal epithelial cell proliferation, as for example inocular epithelial disorders such as epithelial downgrowth or squamouscell carcinomas of the ocular surface.

Yet another aspect of the present invention relates to the use of thesubject methods to control hair growth. Hair is basically composed ofkeratin, a tough and insoluble protein; its chief strength lies in itsdisulphide bond of cystine. Each individual hair comprises a cylindricalshaft and a root, and is contained in a follicle, a flask-likedepression in the skin. The bottom of the follicle contains afinger-like projection termed the papilla, which consists of connectivetissue from which hair grows, and through which blood vessels supply thecells with nourishment. The shaft is the part that extends outwards fromthe skin surface, whilst the root has been described as the buried partof the hair. The base of the root expands into the hair bulb, whichrests upon the papilla. Cells from which the hair is produced grow inthe bulb of the follicle; they are extruded in the form of fibers as thecells proliferate in the follicle. Hair “growth” refers to the formationand elongation of the hair fiber by the dividing cells.

As is well known in the art, the common hair cycle is divided into threestages: anagen, catagen and telogen. During the active phase (anagen),the epidermal stem cells of the dermal papilla divide rapidly. Daughtercells move upward and differentiate to form the concentric layers of thehair itself. The transitional stage, catagen, is marked by the cessationof mitosis of the stem cells in the follicle. The resting stage is knownas telogen, where the hair is retained within the scalp for severalweeks before an emerging new hair developing below it dislodges thetelogen-phase shaft from its follicle. From this model it has becomeclear that the larger the pool of dividing stem cells that differentiateinto hair cells, the more hair growth occurs. Accordingly, methods forincreasing or reducing hair growth can be carried out by potentiating orinhibiting, respectively, the proliferation of these stem cells.

In certain embodiments, the subject methods can be employed as a way ofreducing the growth of human hair as opposed to its conventional removalby cutting, shaving, or depilation. For instance, the present method canbe used in the treatment of trichosis characterized by abnormally rapidor dense growth of hair, e.g. hypertrichosis. In an exemplaryembodiment, subject compounds can be used to manage hirsutism, adisorder marked by abnormal hairiness. The subject method can alsoprovide a process for extending the duration of depilation.

Moreover, because a subject compound will often be cytostatic toepithelial; cells, rather than cytotoxic, such agents can be used toprotect hair follicle cells from cytotoxic agents which requireprogression into S-phase of the cell-cycle for efficacy, e.g.radiation-induced death. Treatment by the subject method can provideprotection by causing the hair follicle cells to become quiescent, e.g.,by inhibiting the cells from entering S phase, and thereby preventingthe follicle cells from undergoing mitotic catastrophe or programmedcell death. For instance, subject compounds can be used for patientsundergoing chemo- or radiation-therapies which ordinarily result in hairloss. By inhibiting cell-cycle progression during such therapies, thesubject treatment can protect hair follicle cells from death which mightotherwise result from activation of cell death programs. After thetherapy has concluded, the instant method can also be removed withconcommitant relief of the inhibition of follicle cell proliferation.

The subject method can also be used in the treatment of folliculitis,such as folliculitis decalvans, folliculitis ulerythematosa reticulataor keloid folliculitis. For example, a cosmetic preparation of a subjectcompound can be applied topically in the treatment ofpseudofolliculitis, a chronic disorder occurring most often in thesubmandibular region of the neck and associated with shaving, thecharacteristic lesions of which are erythematous papules and pustulescontaining buried hairs.

In another aspect of the invention, the subject method can be used toinduce differentiation and/or inhibit proliferation of epitheliallyderived tissue. Such forms of these molecules can provide a basis fordifferentiation therapy for the treatment of hyperplastic and/orneoplastic conditions involving epithelial tissue. For example, suchpreparations can be used for the treatment of cutaneous diseases inwhich there is abnormal proliferation or growth of cells of the skin.

For instance, the pharmaceutical preparations of the invention areintended for the treatment of hyperplastic epidermal conditions, such askeratosis, as well as for the treatment of neoplastic epidermalconditions such as those characterized by a high proliferation rate forvarious skin cancers, as for example basal cell carcinoma or squamouscell carcinoma. The subject method can also be used in the treatment ofautoimmune diseases affecting the skin, in particular, of dermatologicaldiseases involving morbid proliferation and/or keratinization of theepidermis, as for example, caused by psoriasis or atopic dermatosis.

Many common diseases of the skin, such as psoriasis, squamous cellcarcinoma, keratoacanthoma and actinic keratosis are characterized bylocalized abnormal proliferation and growth. For example, in psoriasis,which is characterized by scaly, red, elevated plaques on the skin, thekeratinocytes are known to proliferate much more rapidly than normal andto differentiate less completely.

In one embodiment, the preparations of the present invention aresuitable for the treatment of dermatological ailments linked tokeratinization disorders causing abnormal proliferation of skin cells,which disorders may be marked by either inflammatory or non-inflammatorycomponents. To illustrate, therapeutic preparations of a subjectcompound, e.g., which promotes quiescense or differentiation, can beused to treat varying forms of psoriasis, be they cutaneous, mucosal orungual. Psoriasis, as described above, is typically characterized byepidermal keratinocytes which display marked proliferative activationand differentiation along a “regenerative” pathway. Treatment with anantiproliferative embodiment of the subject method can be used toreverse the pathological epidermal activiation and can provide a basisfor sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatmentwith the subject method. Actinic keratoses, for example, are superficialinflammatory premalignant tumors arising on sun-exposed and irradiatedskin. The lesions are erythematous to brown with variable scaling.Current therapies include excisional and cryosurgery. These treatmentsare painful, however, and often produce cosmetically unacceptablescarring. Accordingly, treatment of keratosis, such as actinickeratosis, can include application, preferably topical, of a subjectcompound composition in amounts sufficient to inhibit hyperproliferationof epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated bythe subject method. Acne vulgaris, for instance, is a multifactorialdisease most commonly occurring in teenagers and young adults, and ischaracterized by the appearance of inflammatory and noninflammatorylesions on the face and upper trunk. The basic defect which gives riseto acne vulgaris is hypercornification of the duct of a hyperactivesebaceous gland. Hypercornification blocks the normal mobility of skinand follicle microorganisms, and in so doing, stimulates the release oflipases by Propinobacterium acnes and Staphylococcus epidermidisbacteria and Pitrosporum ovale, a yeast. Treatment with anantiproliferative subject compound, particularly topical preparations,may be useful for preventing the transitional features of the ducts,e.g. hypercornification, which lead to lesion formation. The subjecttreatment may further include, for example, antibiotics, retinoids andantiandrogens.

The present invention also provides a method for treating various formsof dermatitis. Dermatitis is a descriptive term referring to poorlydemarcated lesions which are either pruritic, erythematous, scaly,blistered, weeping, fissured or crusted. These lesions arise from any ofa wide variety of causes. The most common types of dermatitis areatopic, contact and diaper dermatitis. For instance, seborrheicdermatitis is a chronic, usually pruritic, dermatitis with erythema,dry, moist, or greasy scaling, and yellow crusted patches on variousareas, especially the scalp, with exfoliation of an excessive amount ofdry scales. The subject method can also be used in the treatment ofstasis dermatitis, an often chronic, usually eczematous dermatitis.Actinic dermatitis is dermatitis that due to exposure to actinicradiation such as that from the sun, ultraviolet waves or x- orgamma-radiation. According to the present invention, the subject methodcan be used in the treatment and/or prevention of certain symptoms ofdermatitis caused by unwanted proliferation of epithelial cells. Suchtherapies for these various forms of dermatitis can also include topicaland systemic corticosteroids, antipuritics, and antibiotics.

Ailments which may be treated by the subject method are disordersspecific to non-humans, such as mange.

In still another embodiment, the subject method can be used in thetreatment of human cancers, particularly basal cell carcinomas and othertumors of epithelial tissues such as the skin. For example, subjectcompounds can be employed, in the subject method, as part of a treatmentfor basal cell nevus syndrome (BCNS), and other other human carcinomas,adenocarcinomas, sarcomas and the like.

In another embodiment, the subject method is used as part of a treatmentof prophylaxis regimen for treating (or preventing) basal cellcarcinoma. The deregulation of the Wnt signaling pathway may be ageneral feature of basal cell carcinomas caused by ptc mutations.Consistent overexpression of human ptc mRNA has been described in tumorsof familial and sporadic BCCs, determined by in situ hybridization.Mutations that inactivate ptc may be expected to result inoverexpression of mutant Ptc, because ptc displays negativeautoregulation. Likewise, mutations that inactivate Wnt may be expectedto result in overexpression of mutant Wnt, because Wnt displays negativeautoregulation. Prior research demonstrates that overexpression ofhedgehog proteins can also lead to tumorigenesis. That sonic hedgehog(Shh) has a role in tumorigenesis in the mouse has been suggested byresearch in which transgenic mice overexpressing Shh in the skindeveloped features of BCNS, including multiple BCC-like epidermalproliferations over the entire skin surface, after only a few days ofskin development. A mutation in the Shh human gene from a BCC was alsodescribed; it was suggested that Wnt, Shh or other Hh genes in humanscould act as dominant oncogenes in humans. Sporadic ptc mutations havealso been observed in BCCs from otherwise normal individuals, some ofwhich are UV-signature mutations. In one recent study of sporadic BCCs,five UV-signature type mutations, either CT or CCTT changes, were foundout of fifteen tumors determined to contain ptc mutations. Anotherrecent analysis of sporadic ptc mutations in BCCs and neuroectodermaltumors revealed one CT change in one of three ptc mutations found in theBCCs. See, for example, Goodrich et al., Science 277:1109-13 (1997); Xieet al., Cancer Res. 57:2369-72 (1997); Oro et al., Science 276:817-21(1997); Xie et al., Genes Chromosomes Cancer 18:305-9 (1997); Stone etal., Nature 384:129-34 (1996); and Johnson et al., Science 272:1668-71(1996).

The subject method can also be used to treat patients with BCNS, e.g.,to prevent BCC or other effects of the disease which may be the resultof Wnt-mediated disorders. Basal cell nevus syndrome is a rare autosomaldominant disorder characterized by multiple BCCs that appear at a youngage. BCNS patients are very susceptible to the development of thesetumors; in the second decade of life, large numbers appear, mainly onsun-exposed areas of the skin. This disease also causes a number ofdevelopmental abnormalities, including rib, head and face alterations,and sometimes polydactyly, syndactyly, and spina bifida. They alsodevelop a number of tumor types in addition to BCCs: fibromas of theovaries and heart, cysts of the skin and jaws, and in the centralnervous system, medulloblastomas and meningiomas. The subject methodscan be used to prevent or treat such tumor types in BCNS and non-BCNSpatients. Studies of BCNS patients show that they have both genomic andsporadic mutations in the ptc gene, suggesting that these mutations arethe ultimate cause of this disease.

In another aspect, the present invention provides pharmaceuticalpreparations and methods for controlling the formation ofmegakaryocyte-derived cells and/or controlling the functionalperformance of megakaryocyte-derived cells. For instance, certain of thecompositions disclosed herein may be applied to the treatment orprevention of a variety hyperplastic or neoplastic conditions affectingplatelets.

It will be apparent to one of ordinary skill that certain instancesdescribed above may respond favorably to administration of a Wnt agonistor antagonist, depending on the particular effect on the Wnt pathwaydesired. For example, although a Wnt agonist may be useful inmaintaining a culture of undifferentiated stem cells, a Wnt antagonistmay be employed to maintain a differentiation state in a culture ofdifferentiated cells. Such methods are considered to fall within thescope of the present invention.

In another aspect, the present invention provides pharmaceuticalpreparations comprising the subject aromatic compounds. The aromaticcompounds for use in the subject method may be conveniently formulatedfor administration with a pharmaceutically acceptable and/or sterilemedium, such as water, buffered saline, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like) or suitablemixtures thereof. The optimum concentration of the active ingredient(s)in the chosen medium can be determined empirically, according toprocedures well known to medicinal chemists. As used herein,“biologically acceptable medium” includes any and all solvents,dispersion media, and the like which may be appropriate for the desiredroute of administration of the pharmaceutical preparation. The use ofsuch media for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe activity of the subject compounds, its use in the pharmaceuticalpreparation of the invention is contemplated. Suitable vehicles andtheir formulation inclusive of other proteins are described, forexample, in the book Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA1985). These vehicles include injectable “deposit formulations”.

Pharmaceutical formulations of the present invention can also includeveterinary compositions, e.g., pharmaceutical preparations of thesubject compounds suitable for veterinary uses, e.g., for the treatmentof live stock or domestic animals, e.g., dogs.

Methods of introduction may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a subject compound at a particulartarget site.

The preparations of the present invention may be given orally,parenterally, topically, or rectally. They are of course given by formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, controlled release patch, etc.administration by injection, infusion or inhalation; topical by lotionor ointment; and rectal by suppositories. Oral and topicaladministrations are preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms such as described below orby other conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

In addition to primates, such as humans, a variety of other mammals canbe treated according to the method of the present invention. Forinstance, mammals including, but not limited to, cows, sheep, goats,horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine,canine, feline, rodent or murine species can be treated. However, themethod can also be practiced in other species, such as avian species(e.g., chickens).

The compound of the invention can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with other antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

Embodiments of the present invention also provide articles ofmanufacture that can include a packaging material and a pharmaceuticalcomposition contained within the packaging material. The packagingmaterial can comprise a label which indicates that the pharmaceuticalcomposition can be used for treatment of one or more disordersidentified above.

The pharmaceutical composition can include a compound according to thepresent invention. In addition to a compound of the present invention,the pharmaceutical may also contain other therapeutic agents, and may beformulated, for example, by employing conventional solid or liquidvehicles or diluents, as well as pharmaceutical additives of a typeappropriate to the mode of desired administration (for example,excipients, binders, preservatives, stabilizers, flavors, etc.)according to techniques known in the art of pharmaceutical formulation.

Thus, in one embodiment, the invention provides a pharmaceuticalcomposition including a therapeutic agent and a compound of theinvention. The compound is present in a concentration effective to treatcancer.

The compounds of the invention may be formulated into therapeuticcompositions as natural or salt forms. Pharmaceutically acceptablenon-toxic salts include the base addition salts (formed with freecarboxyl or other anionic groups) which may be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino-ethanol, histidine, procaine, and the like.Such salts may also be formed as acid addition salts with any freecationic groups and will generally be formed with inorganic acids suchas, for example, hydrochloric, sulfuric, or phosphoric acids, or organicacids such as acetic, citric, p-toluenesulfonic, methanesulfonic acid,oxalic, tartaric, mandelic, and the like.

Salts of the invention can include amine salts formed by the protonationof an amino group with inorganic acids such as hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, andthe like. Salts of the invention can also include amine salts formed bythe protonation of an amino group with suitable organic acids, such asp-toluenesulfonic acid, acetic acid, methanesulfonic acid and the like.Additional excipients which are contemplated for use in the practice ofthe present invention are those available to those of ordinary skill inthe art, for example, those found in the United States Pharmacopeia Vol.XXII and National Formulary Vol. XVII, U.S. Pharmacopeia Convention,Inc., Rockville, Md. (1989), the relevant contents of which isincorporated herein by reference. In addition, polymorphs of theinvention compounds are included in the present invention.

Pharmaceutical compositions of the invention may be administered by anysuitable means, for example, orally, such as in the form of tablets,capsules, granules or powders; sublingually; buccally; parenterally,such as by subcutaneous, intravenous, intramuscular, intrathecal, orintracisternal injection or infusion techniques (e.g., as sterileinjectable aqueous or non-aqueous solutions or suspensions); nasallysuch as by inhalation spray; topically, such as in the form of a creamor ointment; or rectally such as in the form of suppositories; in dosageunit formulations containing non-toxic, pharmaceutically acceptablevehicles or diluents. The present compounds may, for example, beadministered in a form suitable for immediate release or extendedrelease. Immediate release or extended release may be achieved by theuse of suitable pharmaceutical compositions comprising the presentcompounds, or, particularly in the case of extended release, by the useof devices such as subcutaneous implants or osmotic pumps. The presentcompounds may also be administered liposomally.

The pharmaceutical compositions for the administration of the compoundsof this embodiment, either alone or in combination with othertherapeutic agents, may conveniently be presented in dosage unit formand may be prepared by any of the methods well known in the art ofpharmacy. All methods include bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general, the pharmaceutical compositions are prepared byuniformly and intimately bringing the active ingredient into associationwith a liquid carrier or a finely divided solid carrier or both, andthen, if necessary, shaping the product into the desired formulation. Inthe pharmaceutical composition the active object compound is included inan amount sufficient to produce the desired effect upon the process orcondition of diseases. The pharmaceutical compositions containing theactive ingredient may be in a form suitable for oral use, for example,as tablets, troches, lozenges, aqueous or oily suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, or syrups orelixirs.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets contain theactive ingredient in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may be for example, inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example, cornstarch, or alginic acid; binding agents, for example starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.Also useful as a solubilizer is polyethylene glycol, for example. Theaqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a parenterally-acceptable diluent or solventor cosolvent or complexing agent or dispersing agent or excipient orcombination thereof, for example 1,3-butanediol, polyethylene glycols,polypropylene glycols, ethanol or other alcohols, povidones, variousbrands of TWEEN surfactant, sodium dodecyl sulfate, sodium deoxycholate,dimethylacetamide, polysorbates, poloxamers, cyclodextrins, lipids, andexcipients such as inorganic salts (e.g., sodium chloride), bufferingagents (e.g., sodium citrate, sodium phosphate), and sugars (e.g.,saccharose and dextrose). Among the acceptable vehicles and solventsthat may be employed are water, dextrose solutions, Ringer's solutionsand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Depending on the condition being treated, these pharmaceuticalcompositions may be formulated and administered systemically or locally.Techniques for formulation and administration may be found in the latestedition of “Remington's Pharmaceutical Sciences” (Mack Publishing Co,Easton Pa.). Suitable routes may, for example, include oral ortransmucosal administration; as well as parenteral delivery, includingintramuscular, subcutaneous, intramedullary, intrathecal,intraventricular, intravenous, intraperitoneal, or intranasaladministration. For injection, the pharmaceutical compositions of theinvention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. For tissue or cellularadministration, penetrants appropriate to the particular barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. Pharmaceutical formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form. Additionally, suspensions of the active compoundsmay be prepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acid esters, such as ethyl oleate or triglycerides,or liposomes. Aqueous injection suspensions may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

The compounds of the present invention may also be administered in theform of suppositories for rectal, urethral, or vaginal administration ofthe drug. These compositions can be prepared by mixing the drug with asuitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Such materials are cocoa butterand polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions,etc., containing the compounds of the present invention are employed.(For purposes of this application, topical application shall includemouthwashes and gargles).

In one embodiment, the invention compounds are administered incombination with an anti-inflammatory agent, antihistamines,chemotherapeutic agent, immunomodulator, therapeutic antibody or aprotein kinase inhibitor, e.g., a tyrosine kinase inhibitor, to asubject in need of such treatment. While not wanting to be limiting,chemotherapeutic agents include antimetabolites, such as methotrexate,DNA cross-linking agents, such as cisplatin/carboplatin; alkylatingagents, such as canbusil; topoisomerase I inhibitors such asdactinomicin; microtubule inhibitors such as taxol (paclitaxol), and thelike. Other chemotherapeutic agents include, for example, a vincaalkaloid, mitomycin-type antibiotic, bleomycin-type antibiotic,antifolate, colchicine, demecoline, etoposide, taxane, anthracyclineantibiotic, doxorubicin, daunorubicin, carminomycin, epirubicin,idarubicin, mithoxanthrone, 4-dimethoxy-daunomycin,11-deoxydaunorubicin, 13-deoxydaunorubicin, adriamycin-14-benzoate,adriamycin-14-octanoate, adriamycin-14-naphthaleneacetate, amsacrine,carmustine, cyclophosphamide, cytarabine, etoposide, lovastatin,melphalan, topetecan, oxalaplatin, chlorambucil, methtrexate, lomustine,thioguanine, asparaginase, vinblastine, vindesine, tamoxifen, ormechlorethamine. While not wanting to be limiting, therapeuticantibodies include antibodies directed against the HER2 protein, such astrastuzumab; antibodies directed against growth factors or growth factorreceptors, such as bevacizumab, which targets vascular endothelialgrowth factor, and OSI-774, which targets epidermal growth factor;antibodies targeting integrin receptors, such as Vitaxin (also known asMEDI-522), and the like. Classes of anticancer agents suitable for usein compositions and methods of the present invention include, but arenot limited to: 1) alkaloids, including, microtubule inhibitors (e.g.,Vincristine, Vinblastine, and Vindesine, etc.), microtubule stabilizers(e.g., Paclitaxel [Taxol], and Docetaxel, Taxotere, etc.), and chromatinfunction inhibitors, including, topoisomerase inhibitors, such as,epipodophyllotoxins (e.g., Etoposide [VP-16], and Teniposide [VM-26],etc.), and agents that target topoisomerase I (e.g., Camptothecin andIsirinotecan [CPT-11], etc.); 2) covalent DNA-binding agents [alkylatingagents], including, nitrogen mustards (e.g., Mechlorethamine,Chlorambucil, Cyclophosphamide, Ifosphamide, and Busulfan [Myleran],etc.), nitrosoureas (e.g., Carmustine, Lomustine, and Semustine, etc.),and other alkylating agents (e.g., Dacarbazine, Hydroxymethylmelamine,Thiotepa, and Mitocycin, etc.); 3) noncovalent DNA-binding agents[antitumor antibiotics], including, nucleic acid inhibitors (e.g.,Dactinomycin [Actinomycin D], etc.), anthracyclines (e.g., Daunorubicin[Daunomycin, and Cerubidine], Doxorubicin [Adriamycin], and Idarubicin[Idamycin], etc.), anthracenediones (e.g., anthracycline analogues, suchas, [Mitoxantrone], etc.), bleomycins (Blenoxane), etc., and plicamycin(Mithramycin), etc.; 4) antimetabolites, including, antifolates (e.g.,Methotrexate, Folex, and Mexate, etc.), purine antimetabolites (e.g.,6-Mercaptopurine [6-MP, Purinethol], 6-Thioguanine [6-TG], Azathioprine,Acyclovir, Ganciclovir, Chlorodeoxyadenosine, 2-Chlorodeoxyadenosine[CdA], and 2′-Deoxycoformycin [Pentostatin], etc.), pyrimidineantagonists (e.g., fluoropyrimidines [e.g., 5-fluorouracil (Adrucil),5-fluorodeoxyuridine (FdUrd) (Floxuridine)] etc.), and cytosinearabinosides (e.g., Cytosar [ara-C] and Fludarabine, etc.); 5) enzymes,including, L-asparaginase; 6) hormones, including, glucocorticoids, suchas, antiestrogens (e.g., Tamoxifen, etc.), nonsteroidal antiandrogens(e.g., Flutamide, etc.), and aromatase inhibitors (e.g., anastrozole[Arimidex], etc.); 7) platinum compounds (e.g., Cisplatin andCarboplatin, etc.); 8) monoclonal antibodies conjugated with anticancerdrugs, toxins, and/or radionuclides, etc.; 9) biological responsemodifiers (e.g., interferons [e.g., IFN-α, etc.] and interleukins [e.g.,IL-2, etc.], etc.); 10) adoptive immunotherapy; 11) hematopoietic growthfactors; 12) agents that induce tumor cell differentiation (e.g.,all-trans-retinoic acid, etc.); 13) gene therapy techniques; 14)antisense therapy techniques; 15) tumor vaccines; 16) therapies directedagainst tumor metastases (e.g., Batimistat, etc.); and 17) inhibitors ofangiogenesis.

The pharmaceutical composition and method of the present invention mayfurther comprise other therapeutically active compounds as noted hereinwhich are usually applied in the treatment of the above mentionedpathological conditions. Examples of other therapeutic agents includethe following: cyclosporins (e.g., cyclosporin A), CTLA4-Ig, antibodiessuch as ICAM-3, anti-IL-2 receptor (Anti-Tac), anti-CD45RB, anti-CD2,anti-CD3 (OKT-3), anti-CD4, anti-CD80, anti-CD86, agents blocking theinteraction between CD40 and gp39, such as antibodies specific for CD40and/or gp39 (i.e., CD154), fusion proteins constructed from CD40 andgp39 (CD40Ig and CD8gp39), inhibitors, such as nuclear translocationinhibitors, of NF-κB function, such as deoxyspergualin (DSG),cholesterol biosynthesis inhibitors such as HMG CoA reductase inhibitors(lovasfatin and simvastatin), non-steroidal antiinflammatory drugs(NSAIDs) such as ibuprofen and cyclooxygenase inhibitors such asrofecoxib, steroids such as prednisone or dexamethasone, gold compounds,antiproliferative agents such as methotrexate, FK506 (tacrolimus,Prograf), mycophenolate mofetil, cytotoxic drugs such as azathioprineand cyclophosphamide, TNF-a inhibitors such as tenidap, anti-TNFantibodies or soluble TNF receptor, and rapamycin (sirolimus orRapamune) or derivatives thereof.

Other agents that may be administered in combination with inventioncompounds include protein therapeutic agents such as cytokines,immunomodulatory agents and antibodies. As used herein the term“cytokine” encompasses chemokines, interleukins, lymphokines, monokines,colony stimulating factors, and receptor associated proteins, andfunctional fragments thereof. As used herein, the term “functionalfragment” refers to a polypeptide or peptide which possesses biologicalfunction or activity that is identified through a defined functionalassay.

The cytokines include endothelial monocyte activating polypeptide II(EMAP-II), granulocyte-macrophage-CSF (GM-CSF), granulocyte-CSF (G-CSF),macrophage-CSF (M-CSF), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, andIL-13, interferons, and the like and which is associated with aparticular biologic, morphologic, or phenotypic alteration in a cell orcell mechanism.

When other therapeutic agents are employed in combination with thecompounds of the present invention they may be used for example inamounts as noted in the Physician Desk Reference (PDR) or as otherwisedetermined by one having ordinary skill in the art.

In the treatment or prevention of conditions which involve cellularproliferation, an appropriate dosage level can generally be betweenabout 0.01 and about 1000 mg per 1 kg of patient body weight per daywhich can be administered in single or multiple doses. For example, thedosage level can be between about 0.01 and about 250 mg/kg per day; morenarrowly, between about 0.5 and about 100 mg/kg per day. A suitabledosage level can be between about 0.01 and about 250 mg/kg per day,between about 0.05 and about 100 mg/kg per day, or between about 0.1 andabout 50 mg/kg per day, or about 1.0 mg/kg per day. For example, withinthis range the dosage can be between about 0.05 and about 0.5 mg/kg perday, or between about 0.5 and about 5 mg/kg per day, or between about 5and about 50 mg/kg per day. For oral administration, the compositionscan be provided in the form of tablets containing between about 1.0 andabout 1,000 mg of the active ingredient, for example, about 1.0, about5.0, about 10.0, about 15.0, about 20.0, about 25.0, about 50.0, about75.0, about 100.0, about 150.0, about 200.0, about 250.0, about 300.0,about 400.0, about 500.0, about 600.0, about 750.0, about 800.0, about900.0, and about 1,000.0 mg of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. The compounds canbe administered on a regimen of 1 to 4 times per day, such as once ortwice per day. There may be a period of no administration followed byanother regimen of administration.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the host undergoing therapy.

Compounds of the present invention can be used, alone or in combinationwith an effective amount of a therapeutic antibody (or therapeuticfragment thereof), a chemotherapeutic or an immunotoxic agent, fortreatment of tumors. Illustrative examples of chemotherapeutic agentsthat can be used for this purpose include doxorubicin, docetaxel, ortaxol. It should be further understood that the invention includescombination therapy including a compound of the invention, including butnot limited to vasculostatic agents, such as tyrosine, serine orthreonine kinase inhibitors, for example, Src-family inhibitors, and anychemotherapeutic agent or therapeutic antibody.

The subject aromatic compounds, and derivatives thereof, can be preparedreadily by employing known synthetic methodology. As is well known inthe art, these coupling reactions are carried out under relatively mildconditions and tolerate a wide range of “spectator” functionality.Additional compounds may be synthesized and tested in a combinatorialfashion, to facilitate the identification of additional compounds whichmay be employed in the subject method.

The aromatic compounds of the present invention, particularly librariesof variants having various representative classes of substituents, areamenable to combinatorial chemistry and other parallel synthesis schemes(see, for example, PCT WO 94/08051). The result is that large librariesof related compounds, e.g. a variegated library of compounds representedabove, can be screened rapidly in high throughput assays in order toidentify potential Wnt regulator lead compounds, as well as to refinethe specificity, toxicity, and/or cytotoxic-kinetic profile of a leadcompound. For instance, Wnt bioactivity assays, such as may be developedusing cells with a Wnt receptor and/or a ptc loss-of-function, hedgehoggain-of-function, or smoothened gain-of-function, can be used to screena library of the subject compounds for those having agonist activitytoward Wnt or antagonist activity towards Wnt. Alternatively,bioactivity assays using cells with Wnt receptors can be used to screena library of the subject compounds for those having antagonist activitytoward Wnt or agonist activity towards Wnt.

Simply for illustration, a combinatorial library for the purposes of thepresent invention is a mixture of chemically related compounds which maybe screened together for a desired property. The preparation of manyrelated compounds in a single reaction greatly reduces and simplifiesthe number of screening processes which need to be carried out.Screening for the appropriate physical properties can be done byconventional methods.

A variety of techniques are available in the art for generatingcombinatorial libraries of small organic molecules such as the subjectaromatic compounds. See, for example, Blondelle et al., Trends Anal.Chem. 14:83 (1995); the Affymax U.S. Pat. Nos. 5,359,115 and 5,362,899:the Ellman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO94/08051; the ArQule U.S. Pat. Nos. 5,736,412 and 5,712,171; Chen etal., JACS 116:2661 (1994); Kerr et al., JACS 115:252 (1993); PCTpublications WO92/10092, WO93/09668 and WO91/07087; and the Lerner etal. PCT publication WO93/20242). Accordingly, a variety of libraries onthe order of about 100 to 1,000,000 or more diversomers of the subjectcompounds can be synthesized and screened for particular activity orproperty.

In an exemplary embodiment, a library of candidate compound diversomerscan be synthesized utilizing a scheme adapted to the techniquesdescribed in the Still et al. PCT publication WO 94/08051, e.g., beinglinked to a polymer bead by a hydrolyzable or photolyzable group,optionally located at one of the positions of the candidate regulatorsor a substituent of a synthetic intermediate. According to the Still etal. technique, the library is synthesized on a set of beads, each beadincluding a set of tags identifying the particular diversomer on thatbead. The bead library can then be “plated” with, for example, cellshaving Wnt receptors for which a Wnt agonist is sought. The diversomerscan be released from the bead, e.g. by hydrolysis.

Many variations on the above and related pathways permit the synthesisof widely diverse libraries of compounds which may be tested asregulators of Wnt function.

There are a variety of assays available for determining the ability ofan aromatic compound such as a Wnt antagonist to regulate Wnt function,many of which can be disposed in high-throughput formats. In many drugscreening programs which test libraries of compounds and naturalextracts, high throughput assays are desirable in order to maximize thenumber of compounds surveyed in a given period of time. Thus, librariesof synthetic and natural products can be sampled for other compoundswhich are Wnt antagonists.

In addition to cell-free assays, test compounds can also be tested incell-based assays. In one embodiment, cells which have a mutationresulting in aberrant cell growth can be contacted with a test agent ofinterest, with the assay scoring for, e.g., inhibition of proliferationof the cell in the presence of the test agent. Such a mutation caninclude, for example, a mutation in the β-catenin gene or a mutation inthe APC gene.

A number of gene products have been implicated in Wnt-mediated signaltransduction, including Wnt, patched, transcription factors of thecubitus interruptus (ci) family, the serine/threonine kinase fused (fu)and the gene products of costal-2, smoothened and suppressor of fused.

The induction of cells by Wnt proteins sets in motion a cascadeinvolving the activation and inhibition of downstream effectors, theultimate consequence of which is, in some instances, a detectable changein the transcription or translation of a gene. Potential transcriptionaltargets of Wnt-mediated signaling are the GSK3β complex, and thefrizzled receptor (Fz). By selecting transcriptional regulatorysequences from such target genes, that are responsible for the up- ordown-regulation of these genes in response to Wnt signaling, andoperatively linking such promoters to a reporter gene, one can derive atranscription based assay which is sensitive to the ability of aspecific test compound to modify Wnt-mediated signaling pathways.Expression of the reporter gene, thus, provides a valuable screeningtool for the development of compounds that act as antagonists to Wnt.

Reporter gene based assays of this invention measure the end stage ofthe above described cascade of events, e.g., transcriptional modulation.Accordingly, in practicing one embodiment of the assay, a reporter geneconstruct is inserted into the reagent cell in order to generate adetection signal dependent on stimulation by Wnt. The amount oftranscription from the reporter gene may be measured using any methodknown to those of skill in the art to be suitable. For example, mRNAexpression from the reporter gene may be detected using RNAse protectionor RNA-based PCR, or the protein product of the reporter gene may beidentified by a characteristic stain or an intrinsic biologicalactivity. The amount of expression from the reporter gene is thencompared to the amount of expression in either the same cell in theabsence of the test compound or it may be compared with the amount oftranscription in a substantially identical cell that lacks the targetreceptor protein. Any statistically or otherwise significant decrease inthe amount of transcription indicates that the test compound has in somemanner antagonized the normal Wnt signal, e.g., the test compound is apotential Wnt antagonist.

A particular advantage to the screening assays of the invention findsapplication to the design of personalized medicine. For example, aplurality of test agents can be arranged in an array, which can be anaddressable array, on a solid support such as a microchip, on a glassslide, on a bead, or in a well, and the cells of a subject (e.g., abiopsy sample) can be contacted with the different test agents toidentify one or more agents having desirable characteristics, including,for example, in addition to the ability to modulate Wnt activity,minimal or no toxicity to the cell, desirable solubilitycharacteristics, and the like. Consequently, a treatment regimen may betailored specifically to the individual based upon the subject's levelsof Wnt activity, Fz activity, and/or GSK3β activity.

Once disease is established and a treatment protocol is initiated,screening assays of the invention may be repeated on a regular basis toevaluate whether any of the levels of Wnt activity, Fz activity, and/orGSK3β activity in the patient begins to approximate that which isobserved in a normal patient. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to months. Accordingly, the invention is alsodirected to methods for monitoring a therapeutic regimen for treating asubject having cancer. A comparison of any of the levels of Wntactivity, Fz activity, and/or GSK3β activity prior to and during therapyindicates the efficacy of the therapy. Therefore, one skilled in the artwill be able to recognize and adjust the therapeutic approach as needed.

As used herein, a “corresponding normal sample” is any sample taken froma subject of similar species that is considered healthy or otherwise notsuffering from a cancer disease being treated. As such, anormal/standard levels of Wnt activity, Fz activity, and/or GSK3βactivity denotes the level of Wnt activity, Fz activity, and/or GSK3βactivity present in a sample from the normal sample. A normal level ofWnt activity, Fz activity, and/or GSK3β activity can be established bycombining body fluids or cell extracts taken from normal healthysubjects, preferably human, with antibody to the proteins of interestunder conditions suitable for Wnt activity, Fz activity, and/or GSK3βactivity. Levels of Wnt activity, Fz activity, and/or GSK3β activity insubject, control, and disease samples from biopsied tissues can becompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing and treatingdisease. A normal level of Wnt activity, Fz activity, and/or GSK3βactivity also can be determined as an average value taken from apopulation of subjects that is considered to be healthy, or is at leastfree of cancer. A variety of protocols including ELISA, RIA, and FACSare useful for measuring levels of Wnt activity, Fz activity, and/orGSK3β activity, and provide a basis for diagnosing altered or abnormallevels Wnt activity, Fz activity, and/or GSK3β activity.

The following examples are provided to further illustrate the advantagesand features of the present invention, but are not intended to limit thescope of the invention.

EXAMPLE 1 Screen for Small-Molecule Modulators of the Wnt SignalingPathway

A Wnt-sensitive reporter (7xTCF-luciferase (Firefly) (pSUPERTOPFLASH)and constitutively expressed control reporter (Renilla luciferasepRL-SV40, Promega) were transfected into the indicated cell lines (FIG.1), and cells were stimulated with Wnt3A-containing medium as a sourceof activating ligand (medium conditioned by growth of Wnt3A/L cells;CRL-2647, ATCC). Wnt-sensitive reporter activity, normalized to theconstitutive reporter, was induced by as much as several hundred fold ascompared to basal levels.

Taking advantage of sensitive, synthetic TCF/LEF site-based promotersfused to the firefly luciferase gene (e.g., pSUPERTOPFLASH; Lum et al.,2003) and various Wnt-responsive mammalian cell lines we engineeredstable, cell-based reporter systems useful for high-throughput screensof Wnt pathway modulators. These lines also feature Renilla luciferasecontrol reporters (e.g., pRL-SV40; Promega) that are stably integratedand constitutively expressed. Using Wnt3A-conditioned medium as a sourceof activating ligand (see CRL-2647, ATCC) we are able to routinelyachieve induced reporter activities measuring 100- to 200-fold higherthan basal levels (J. Chen, R. Mann, P. Beachy, unpublished). The assaysystem easily scaled-down to a 384-well format and, using pin transferson Biomek FX robotics (Beckman), we screened 40,000 compounds fromChemDiv and ChemBridge collections (at a concentration of approx. 2micromolar) and isolated small sets of candidate Wnt pathway agonistsand antagonists (unpublished). Secondary screens using luciferasereporters that are constitutively expressed enabled identification ofcompounds that act directly on the assay reporter enzyme rather thanthrough modulation of transcription through TCF sites. Dose-responseexperiments were used to calculate IC₅₀s of candidate compounds; theprofile of one of the candidate Wnt antagonists (A6) is shown in FIG. 2.

Dose-response behavior of the Wnt reporter (7xTCF-luciferase (Firefly)(pSUPERTOPFLASH) and control reporter (Renilla luciferase (pRL-SV40))with candidate Wnt antagonist A6 (number 4687-0060) is shown in FIG. 2.At 5 μM there is a marked reduction in luciferase activity with only aminor effect on control Renilla luciferase. The IC₅₀ of A6 is estimatedto be 2-3 μM. Toxicity, as indicated by a dramatic loss of both Fireflyluciferase and Renilla luciferase levels, was observed at the highestconcentration (50 μM) used in the titration experiment (not shown). At 5μM there was no effect on the Firefly luciferase activity derived from aconstitutive promoter (not shown).

The screen for compounds that modulate Wnt pathway activity alsouncovered an agonist for the Wnt pathway, i.e., a compound thatstimulates expression of the Wnt reporter in the absence of Wnt proteinstimulation. Preliminary studies suggest that this Wnt pathway agonistacts directly on the transcriptional machinery of this signalingcascade. Normal pathway activation induced by Wnt proteins results inthe stabilization and cellular accumulation of a protein calledβ-catenin. This increase in β-catenin levels promotes the conversion ofTCF/LEF repressors to transcriptional activators, thereby inducing theexpression of Wnt target genes. In contrast, the small molecule agonistactivates the Wnt pathway without causing β-catenin stabilization,suggesting that it modulates TCF/LEF activity in a more direct manner(FIG. 3, A and B). This model is supported by our recent observationthat the subcellular localization of LEF-1 changes upon the addition ofthe synthetic agonist to Wnt-responsive cells (FIG. 3, C and D). Thiscompound or more potent derivatives may be useful for stimulation of theWnt pathway for protection from injury or to stimulate recovery fromtissue injury, in a variety of tissues ranging from the nervous systemto the hematopoietic system.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A compound having structure (I):

or a pharmaceutically acceptable salt thereof.
 2. A compound havingstructure (II):

or a pharmaceutically acceptable salt thereof.
 3. A compound havingstructure (III):

or a pharmaceutically acceptable salt thereof.
 4. A compound, comprisinga first aromatic moiety fused to a second aromatic moiety, wherein thefirst aromatic moiety is naphthalene-1,4-dione group and the secondaromatic moiety is an N-substituted triazole-N-oxide group, or apharmaceutically acceptable salt thereof.
 5. The compound of claim 4,wherein the substitutent in the N-substituted triazole-N-oxide moietycomprises an alkylaryl group.
 6. The compound of claim 5, wherein thealkylaryl group is benzyl group.
 7. A compound comprising abenzopiperazine moiety fused to a substituted imidazole moiety, or apharmaceutically acceptable salt thereof.
 8. The compound of claim 7,wherein the benzopiperazine moiety includes an alkylpiperazinyl group.9. The compound of claim 8, wherein the alkylpiperazinyl group ismethylpirazinyl group.
 10. The compound of claim 7, wherein theimidazole moiety comprises a phenyl substitutent.
 11. The compound ofclaim 10, wherein the imidazole moiety further includes a halogenatedaromatic group attached to a nitrogen atom in the imidazole structure.12. The compound of claim 11, where the halogenated aromatic group is abromophenyl group.
 13. A compound, comprising an azaanthracene moietyand a secondary amino moiety, or a pharmaceutically acceptable saltthereof.
 14. The compound of claim 13, wherein the secondary aminomoiety is attached to the nitrogen-containing ring of the azaanthracenemoiety.
 15. A method for treating a cell proliferative disorder in asubject, said method comprising administering an effective amount of thecompound of claim 1, or pharmaceutically acceptable salts, hydrates,solvates, crystal forms and individual diastereomers thereof, to asubject in need of such treatment.
 16. The method of claim 15, whereinthe cell proliferative disorder is selected from a group consisting ofbasal cell carcinoma, medulloblastoma and meningioma.
 17. The method ofclaim 15, wherein the subject is a human or another mammal.
 18. Themethod of claim 15, further including administering the compound incombination with a therapeutic agent, immunomodulatory agent,therapeutic antibody or an enzyme inhibitor.
 19. The method of claim 18,wherein the therapeutic agent is selected from a group consisting ofmethotrexate, cisplatin/carboplatin, canbusil, dactinomicin, taxol(paclitaxel), antifolate, colchicine, demecoline, etoposide,taxane/taxol, docetaxel, doxorubicin, anthracycline antibiotic,doxorubicin, daunorubicin, carminomycin, epirubicin, idarubicin,mithoxanthrone, 4-dimethoxy-daunomycin, 11-deoxydaunorubicin,13-deoxydaunorubicin, adriamycin-14-benzoate, adriamycin-14-octanoate oradriamycin-14-naphthaleneacetate, irinotecan, topotecan, gemcitabine,5-fluorouracil, leucovorin carboplatin, cisplatin, taxanes,tezacitabine, cyclophosphamide, vinca alkaloids, imatinib,anthracyclines, rituximab, trastuzumab, bevacizumab, OSI-774, andVitaxin.
 20. A pharmaceutical composition comprising the compound ofclaim 1, in a pharmaceutically acceptable carrier.
 21. An article ofmanufacture comprising packaging material and a pharmaceuticalcomposition contained within the packaging material, wherein thepackaging material comprises a label which indicates that thepharmaceutical composition can be used for treatment of disorders andwherein said pharmaceutical composition comprises the compound ofclaim
 1. 22. A process for making a pharmaceutical compositioncomprising the compound of claim 1, or its pharmaceutically acceptablesalts, hydrates, solvates, crystal forms salts and individualdiastereomers thereof, and a pharmaceutically acceptable carrier.
 23. Amethod of inhibiting an altered growth state of a cell having a Wntreceptor, comprising contacting the cell with a composition comprisingthe compound of claim
 1. 24. The method of claim 23, wherein thecompound is a Wnt signal transduction agonist.
 25. The method of claim24, wherein the agonist agonizes Fz inhibition of Wnt signaling.
 26. Themethod of claim 24, wherein the agonist agonizes GSK3β inhibition of Wntsignaling.
 27. The method of claim 23, wherein the compound is a Wntsignal transduction antagonist.
 28. The method of claim 27, wherein theantagonist antagonizes Fz inhibition of Wnt signaling.
 29. The method ofclaim 27, wherein the antagonist antagonizes GSK3β inhibition of Wntsignaling.
 30. The method of claim 27, wherein the antagonist interfereswith activation of a Wnt-mediated signal transduction pathway.
 31. Themethod of claim 23, wherein the cells are normal cells.
 32. The methodof claim 23, wherein the cells are cancer cells.
 33. The method of claim23, wherein the contacting is performed in vivo.
 34. The method of claim23, wherein the contacting is performed in vitro.
 35. The method ofclaim 23, wherein the composition is administered as part of atherapeutic or cosmetic application.
 36. The method of claim 35, whereinthe therapeutic or cosmetic application is regulation of neural tissues,bone and cartilage formation and repair, regulation of spermatogenesis,regulation of smooth muscle, regulation of lung, liver and other organsarising from the primitive gut, regulation of hematopoietic function, orregulation of skin and hair growth.
 37. A method of identifying acompound that modulates cell proliferation in a cell having a Wntreceptor, a Fz receptor or a GSK3β receptor, comprising: a) incubatingcomponents comprising the compound of claim 1, a test compound, and acell having a Wnt receptor, a Fz receptor or a GSK3β receptor, underconditions sufficient to allow the components to interact; and b)measuring the ability of the test compound to affect cell proliferationby detecting an increase or decrease in expression of signaltransduction activity.
 38. The method of claim 37, wherein the signaltransduction activity is expression of Wnt.
 39. A method for inhibitingthe growth of a tumor cell in a subject in need thereof, comprisingadministering to a tumor cell an effective amount of the compound ofclaim
 1. 40. A method of monitoring a therapeutic regimen for treating asubject having a cell proliferative disorder comprising determining achange in cell proliferation during therapy.
 41. The method of claim 40,wherein the therapy comprises the treatment of claim 15.